Cellartis® Cardiomyocytes User Manual
Contents
1. Add 0 25 96 trypsin EDTA to each well 100 ul cm and incubate for 2 4 min Gently detach the cells by dispensing the dissociation solution over the surface using a 1 ml pipette Add one volume 100 ul cm of Cellartis CM Culture Medium with 5 uM Y 27632 to each well to deactivate the trypsin Transfer the cell suspension into a suitable tube NU Ee p Count the cardiomyocytes Centrifuge the cells at 200 x g for 5 min at RT 10 Aspirate the supernatant and gently resuspend the cell pellet in an appropriate volume of Cellartis CM Culture Medium with 5 uM Y 27632 to the desired concentration 11 Seed the cells at the desired density using 0 4 0 6 ml cm Cellartis CM Culture Medium with 5 uM Y eo oN 27632 Contact Us Customer Service Ordering Technical Support tel 46 0 31 758 0900 tel 46 0 31 758 0900 fax 46 0 31 758 0910 fax 46 0 31 758 0910 web www clontech com web www clontech com e mail info cellartis takara clontech eu e mail tech cellartis takara clontech eu Notice to Purchaser This product is for research use only It is not intended for use in therapeutic or diagnostic procedures for humans or animals Also do not use this product as food cosmetic or household item etc This product may not be resold or transferred modified for resale or transfer or used to manufacture commercial products without written approval from Takara Bio Europe AB If you req2. RR oL TER ALT e pud LOS 4 VII Thawing of Cellartis Cardiomyocytes esessseeeseeeseeeseeen eene enne nnen nennen nnne tnne tenete enne enr enr en nennen nenne 5 PME IER TT Pm 5 Be Thawing Ces E a 5 IX Medium Change of Cellartis Cardiomyocytes esses enne rennen rennen enses 5 Preparati 5 B Medium Change Day l 4 ntf eei cei E E R 5 C Medium Change Day 3 Onwards esses enne enne neen nens tnee tnnt tne sne teneret rennen enses 6 X Re seeding of Cellartis Cardiomyocytes in Application Formats eese 6 Table of Figures Figure 1 Schematic presentation of the Cellartis Cardiomyocyte workflow eese rene 4 061215 www clontech com Page 2 of 6 Takara Bio Europe AB A Takara Bio Company Cellartis amp Cardiomyocytes User Manual Introduction Cellartis Cardiomyocytes are human cardiomyocytes derived from a human induced pluripotent stem iPS cell line The iPS cells have been differentiated into spontaneously beating cardiomyocytes in vitro The cells have subsequently been dissociated into a single cell suspension and frozen in vials Cellartis Cardiomyocytes are provided with basal media for thawing and maintenance FBS needs to be added to the media before use This product should be handled only by persons who have been trained in laboratory techniques and in acc
3. Add 10 ml FBS per 90 ml Cellartis CM Culture Base to achieve Cellartis CM Culture Medium 4 Cellartis CM Culture Medium should be stored at 4 C and expires one month after the date of preparation 5 Always discard any leftover warmed Cellartis CM Culture Medium 061215 www clontech com Page 4 of 6 Takara Bio Europe AB A Takara Bio Company Cellartis amp Cardiomyocytes User Manual Vill Thawing of Cellartis Cardiomyocytes It is recommended that a maximum of two to three vials are thawed at one time The recommended seeding density is 1 2 1 5 x 10 viable cells cm A Preparation e Prepare the appropriate volume of Cellartis CM Thawing Medium with Y 27632 by adding Y 27632 to a final concentration of 10 uM Warm to room temperature RT 15 25 C e Coat the appropriate number of cell culture vessels according to section VI Thawing Cells NOTE For your protection wear a protective face mask and protective gloves Use forceps when handling a frozen vial Never hold the vial in your hand as the cryovial may explode due to rapid temperature changes 1 Transfer as quickly as possible the frozen vials from liquid nitrogen to a 37 C 1 C water bath using forceps 2 Thaw the cells by gently pushing the vial under the surface of the water without swirling the vial Do not submerge the cap of the vial in the water bath as this could contaminate the cells 3 Take the vials out of the water bath as soon as the thawing is compl
4. Takara Bio Europe AB Cellartis Cardiomyocytes User Manual Cat No Y10075 061215 Takara Bio Europe AB A Takara Bio Company Arvid Wallgrens backe 20 SE 413 46 Goteborg Sweden Europe Technical Support tech cellartis takara clontech eu United States Canada Asia Pacific Europe Japan Page 1 of 6 800 662 2566 1 650 919 7300 46 0 31 758 0900 amp 81 0 77 543 6116 Cellartis amp Cardiomyocytes User Manual Table of Contents I Uitte OQu Ct omnis E Dn 3 IH Listof Components ceo doo Cr penne o eig na IEEE E ed B itae ih Po lan engines 3 III Additional Material Required eei terree seen e peat reta Lei eo eese arta dpa EXER seas KEVER ENATS Pu a bates spesueds 3 IV General considerations nate ee irse rrt eate iere LEE e ELA EE Doe eet E E Heres ba Eo DR Ree ET ae ea OPER e Leu bur 3 As Storage and Handling oett nee tr ERE e e c re HE Ora rcu ER RECEN E aTe 3 V Culture of Cellartis Cardiomyocytes esses enne enne nnnen nest neentnee tree tnnt ttes trente tnr enn rennen nennen nenne 4 VI Coating of Cell Culture Vessels cia diter Hop ele ep HL ete Roe ERR EE ARTELE e Eod te o eda eb D eder 4 VIL Cellartis CM Medium Preparation esses eene nennen ea a thes nennen tenete tenete ener enne 4 A Cellartis CM Thawimg Me Qin vis onini erts beret et heise Ping pr see bo Nope EE oec ve eva E NEEE 4 B Cellartis CM Culture Medium on ERG FREE es HE ea eL eee eL HERE AE RR ROM ERE
5. dium as described in section VII B Warm to 37 C 1 C B Medium Change Day 1 NOTE Work very gently at this point in order not to detach the cells 1 Wash the cells carefully by aspirating the Cellartis CM Thawing Medium and gently flush the cells with the medium two to three times in order to remove non attached cells 2 Aspirate the old medium and add warm Cellartis CM Culture Medium 0 4 0 6 ml cm 061215 www clontech com Page 5 of 6 Takara Bio Europe AB A Takara Bio Company Cellartis amp Cardiomyocytes User Manual 3 Return the cell culture unit to an incubator with 37 C 1 C 5 CO and gt 90 humidity C Medium Change Day 3 Onwards 1 Gently aspirate the entire volume of medium from the culture vessel and discard 2 Add warm Cellartis CM Culture Medium 0 4 0 6 ml cm 3 Return the cell culture vessel to the incubator with 37 C 1 C 5 CO and gt 90 humidity X Re seeding of Cellartis Cardiomyocytes in Application Formats Seeding density and coating are dependent on the intended application and may require optimization A recommended starting point is to seed 1 2 1 5 x 10 viable cells cm on culture vessels coated with 7 3 ug fibronectin cm 1 Warm an appropriate amount of Cellartis CM Culture Medium to 37 C 1 C Add Y 27632 to a final concentration of 5 uM prior to use Aspirate the medium from each well containing cardiomyocytes Rinse the wells with D PBS using approximately 0 25 ml cm
6. eted approximately 3 min the vials should still be cold on the outside Wipe the vials with an appropriate disinfectant and place into the biological safety cabinet Scop As soon as possible gently transfer the cell suspension into a sterile 50 ml tube by using a pipette 6 Rinse the vial with 1 ml of Cellartis CM Thawing Medium with 10 uM Y 27632 and carefully add it to the cell suspension dropwise 7 Add 8 ml of Cellartis CM Thawing Medium with 10 uM Y 27632 dropwise Gently swirl the tube a few times in between 8 Centrifuge the tube at 200 x g for 5 min at RT and remove the supernatant 9 Carefully resuspend the cell pellet with Cellartis CM Thawing Medium with 10 uM Y 27632 using 6 ml medium per thawed vial 10 Count the cells and measure viability 11 Add Cellartis CM Thawing Medium with 10 uM Y 27632 to the desired cell density and seed the cells using 0 4 0 6 ml cm Make sure the entire surface is covered with medium 12 Transfer the cell culture unit to an incubator at 37 C 1 C 5 CO and gt 90 humidity and leave untouched for 24 hr NOTE The cells can be used starting at day three after thawing two days after the first medium change IX Medium Change of Cellartis Cardiomyocytes At day one after thawing the non attached cells are washed away and the medium is changed After day one the medium should be changed every second to third day A Preparation e Prepare Cellartis CM Culture Me
7. hawing the cells can be dissociated and moved to the preferred culture assay format see dissociation protocol in section X Day 0 Day 1 Day 2 Day 3 Thaw amp Wash amp Change Change Plate VIII Medium IX B Medium IX C Figure 1 The Cellartis Cardiomyocyte workflow Corresponding sections of this user manual are referenced in parentheses VI Coating of Cell Culture Vessels 1 Dilute the required volume of fibronectin in D PBS final concentration 50 ug ml 2 Add the fibronectin solution to the cell culture vessels 0 3 ml cm corresponding to 15 ug cm Make sure the entire surface is covered 3 Incubate at 37 C 1 C and 596 CO for 23 hr 4 Remove the fibronectin solution from the cell culture vessels just before use Vil Cellartis CM Medium Preparation A Cellartis CM Thawing Medium 1 Thaw Cellartis CM Thawing Base 2 Decontaminate the external surface of all bottles with an appropriate disinfectant and place into the biological safety cabinet 3 Add 8 ml FBS per 32 ml Cellartis CM Thawing Base to achieve Cellartis CM Thawing Medium 4 Cellartis CM Thawing Medium should be stored at 4 C and expires one month after the date of preparation 5 Always discard any leftover warmed Cellartis CM Thawing Medium B Cellartis CM Culture Medium 1 Thaw Cellartis CM Culture Base 2 Decontaminate the external surface of supplement and medium bottle with appropriate disinfectant and place into the biological safety cabinet 3
8. ordance with the principles of good cell culture practice Takara Bio Europe AB recommends the use of media and reagents according to this manual for optimal performance of the cells Takara Bio Europe AB cannot guarantee correct technical feedback on customer cultures unless the below culture instructions have been followed List of Components e Cellartis Cardiomyocytes from ChiPSC22 Kit Cat No Y10075 o 1 vial Cellartis Cardiomyocytes from ChiPSC22 Cat No Y10076 not sold separately 53 x 106 viable cells o l bottle Cellartis CM Thawing Base Cat No Y10062 32 ml o 1 bottle Cellartis CM Culture Base Cat No Y10063 90 ml Additional Material Required The following materials are required but not supplied e Fetal Bovine Serum FBS Life Technologies Cat No 16140 e Fibronectin Sigma Aldrich Cat No F0895 e PBS Dulbecco s with Ca amp Mg D PBS e PBS Dulbecco s w o Ca amp Mg D PBS e Trypsin EDTA 0 25 phenol red e Y 27632 e Cell culture vessels tissue culture treated polystyrene surface e General cell culture equipment used in cell culture laboratory Only needed if dissociating Cellartis Cardiomyocytes see section X General considerations A Storage and Handling Cellartis Cardiomyocytes should be stored at lt 150 C Under recommended storage conditions the cells can be stored for up to one year from date of receipt NOTE When transferring the cells from the transport
9. uire licenses for other use please contact us by phone at 46 31 758 0900 Your use of this product is also subject to compliance with any applicable licensing requirements as detailed in our catalogues on our website at http www clontech com on the label or other documentation accompanying the goods It is your responsibility to review understand and adhere to any restrictions imposed by such statements Takara and the Takara logo are trademarks of TAKARA HOLDINGS INC Kyoto Japan Cellartis is a trademark of Takara Bio Europe AB All other trademarks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Takara Bio Europe AB is a Takara Bio company 2015 Takara Bio Europe AB This document has been reviewed and approved by the Takara Bio Europe AB Quality Assurance Department 061215 www clontech com Page 6 of 6 Takara Bio Europe AB A Takara Bio Company
10. vessel to long term storage immediate transfer is essential since variations in temperature may have an adverse effect on cell survival and quality Cellartis Cardiomyocytes should be maintained in an incubator at 37 C 1 C 5 CO and gt 90 humidity Cellartis CM Thawing Base and Cellartis CM Culture Base should be stored at 20 C The expiration date is indicated on the label Complete Cellartis CM Thawing Medium and Cellartis CM Culture Medium should be prepared as described in section VII 061215 www clontech com Page 3 of 6 Takara Bio Europe AB A Takara Bio Company Cellartis amp Cardiomyocytes User Manual V Culture of Cellartis Cardiomyocytes Cellartis Cardiomyocytes are thawed and plated in Cellartis CM Thawing Medium The day after thawing the cultures are washed to remove non attached cells and the medium is changed to Cellartis Culture Medium The cardiomyocytes can be used starting at day three after thawing two days after the first medium change Medium should be changed every second to third day The cardiomyocytes can be maintained in culture for at least 14 days after thawing under the recommended conditions The workflow is depicted in Figure 1 For applications where non standard culture formats are used it is recommended that the cells are thawed and plated in 6 or 12 well tissue culture treated plates coated according to section VI for optimal recovery When the cells have recovered day three after t
Download Pdf Manuals
Related Search